The subject invention is directed generally to gamma glutamyl hydrolase, and more particularly to the identification of the active site within gamma glutamyl hydrolase.
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
Many enzymes involved in producing precursors for DNA synthesis require folate as a cofactor. Antifolate drugs which impair folate function, such as methotrexate (MTX), are the primary treatments for many cancers. The retention and efficacy of folates and antifolate drugs within the cell are dependent on the addition of a poly-xcex3-glutamate chain to the monoglutamate (McBurney and Whitmore 1974). Folylpolyglutamate synthetase (FPGS) catalyzes the sequential addition of glutamate (for reviews see McGuire and Coward 1984; Shane 1989) and xcex3-glutamyl hydrolase (GH, EC 3.4.19.9) catalyzes the removal of glutamate from folyl- and antifolylpoly-xcex3-glutamates (for review see Galivan and Ryan 1998). In conjunction with the folate transport systems, the balance between GH and FPGS activity regulates the amount of glutamylation of folate and antifolate drugs in the cell.
GH activity in mammalian cells is found in the lysosomes (McGuire and Coward 1984; Hoffbrand and Peters 1969; Silink and Rowe 1975a; Wang et al. 1986; Yao et al. 1995). However, in cell culture, the major part of the synthesized enzyme is secreted into the medium (O""Connor et al. 1991). In humans, GH activity has been detected in plasma (Baggott et al. 1987), bile (Horne et al. 1981), pancreatic juice (Bhandari et al. 1990), and jejunal mucosa (Reisenauer et al. 1977). The enzyme has been purified from a number of mammalian tissues (Saini and Rosenberg 1974; Silink et al. 1975b; Rao and Norohna 1977; Elsenhans et al. 1984; Wang et al. 1993) and cell lines (Yao et al. 1995; O""Connor et al. 1991; Wang et al. 1993; Rhee et al. 1998). Both rat GH and human GH are glycoproteins (Rhee et al. 1998; Yao et al. 1996a). GH from different species has a different specificity in the hydrolysis of the poly-xcex3-glutamyl tail. For example, the rat GH enzyme acts as an endopeptidase (Wang et al. 1993) hydrolyzing the innermost xcex3-glutamyl bond and releasing the poly-xcex3-glutamate chain as a single unit. Conversely GH isolated from human sources (hGH) removes only the carboxyl terminal glutamate or di-xcex3-glutamate during the reaction (Rhee et al. 1998).
The cDNA""s encoding GH from rat and human sources have been isolated (Yao et al. 1996a; Yao et al. 1996b; U.S. Pat. No. 5,801,031, issued Sep. 1, 1998 and incorporated herein by reference) and a mouse GH cDNA has recently been isolated (Esaki et al. 1998). The hGH cDNA has been expressed in both an insect expression system (Rhee et al. 1998) and Escherichia coli (Yao et al. 1996b). The first 24 amino acids encoded by the hGH cDNA are a signal peptide, which is removed during processing (Rhee et al. 1998). Therefore, the N-terminal amino acid of the mature hGH enzyme is equivalent to R25 in the published hGH sequence (Yao et al. 1996b).
Early studies demonstrated that GH is sulfhydryl sensitive and is inhibited by iodoacetic acid or p-hydroxymercuribenzoate (pHMB) (McGuire and Coward 1984; Reisenauer et al. 1977; Silink et al. 1975b). Studies on GH in lysosomes isolated from murine S180 cells indicate that accumulation of reduced sulfhydryls in the lysosome activate GH (Barrueco et al. 1992). Recent studies with pure GH preparations verified the earlier findings of sulfhydryl sensitivity (O""Connor et al. 1991; Rhee et al. 1998; Yao et al. 1996a).
The catalytic mechanism of GH has yet to be defined. A better understanding of the mechanism could lead to the specific inhibition of this enzyme and increased efficacy of antifolate drugs.
The subject invention addresses this need by identifying the active site of GH as including amino acid residues 110, 171, 220 and 222 of SEQ ID NO:1. SEQ ID NO:1 represents the amino acid sequence of mature human native GH (without the signal peptide). The subject invention thus provides an inactive gamma glutamyl hydrolase protein, the inactive protein having an amino acid sequence that substantially corresponds to the amino acid sequence of native gamma glutamyl hydrolase as shown in SEQ ID NO:1, SEQ ID NO:1 being modified at one or more of amino acid residues 110, 171, 220 or 222 to render the resulting gamma glutamyl hydrolase protein inactive. The invention further provides a fragment of the inactive gamma glutamyl hydrolase protein, wherein the fragment is from about 10 to about 150 amino acids in length and wherein the fragment includes one or more of the modified amino acid residues.
Also provided by the subject invention is a method of inactivating a gamma glutamyl hydrolase protein. The method comprises: providing a gamma glutamyl hydrolase protein; and modifying one or more of amino acid residues 110, 171, 220 or 222 in the amino acid sequence of the gamma glutamyl hydrolase protein as shown in SEQ ID NO:1, thereby inactivating the gamma glutamyl hydrolase protein.
Further provided is a molecule capable of binding to one or more of amino acid residues 110, 171, 220 or 222 in the amino acid sequence of gamma glutamyl hydrolase as shown in SEQ ID NO:1, wherein the molecule inactivates gamma glutamyl hydrolase and wherein the molecule has a three dimensional structure complementary to the three dimensional structure of gamma glutamyl hydrolase in a fragment that includes one or more of the one or more of amino acid residues 110, 171, 220 or 222. Compositions comprising the molecule and a suitable carrier, and the molecule and an antifolate, are also provided. The molecule can be used with an antifolate to increase the effectiveness of antifolate treatment.
The molecule can also be used to inactivate gamma glutamyl hydrolase protein. Such a method comprises: providing a gamma glutamyl hydrolase protein; and exposing the gamma glutamyl hydrolase protein to the above-described molecule, wherein the molecule binds to one or more of amino acid residues 110, 171, 220 or 222 in the amino acid sequence of the gamma glutamyl hydrolase protein thereby inactivating the gamma glutamyl hydrolase protein.
Further provided is a method of identifying a molecule that inactivates gamma glutamyl hydrolase protein. The method comprises: determining whether a molecule binds to one or more of amino acid residues 110,171, 220 or 222 in the amino acid sequence of gamma glutamyl hydrolase as shown in SEQ ID NO:1; and screening a molecule that binds to one or more of amino acid residues 110, 171, 220 or 222 to determine whether the screened molecule inactivates gamma glutamyl hydrolase protein. A molecule identified by the method, as well as a method of inactivating gamma glutamyl hydrolase using the identified molecule, are also provided.
Further provided is a nucleic acid molecule encoding an inactive gamma glutamyl hydrolase protein, the nucleic acid molecule encoding an amino acid sequence that substantially corresponds to the amino acid sequence of native gamma glutamyl hydrolase as shown in SEQ ID NO:1, SEQ ID NO:1 being modified at one or more of amino acid residues 110, 171, 220 or 222 to render the resulting gamma glutamyl hydrolase protein inactive.